Huntington's disease (HD) is the most prevalent autosomal dominant, trinucleotide repeat neurodegenerative disease. The huntingtin gene encodes a protein of 350 kD; the disease causing mutation is an expansion of an amino-terminal polyglutamine repeat of more than 36 successive glutamines. Our broad research goal is to understand the molecular basis of HD pathogenesis to target therapy. Because HD is an inherited disease, we expected that the mutant allele will differ from wild-type by at least a single nucleotide polymorphism, thereby offering a target for gene silencing by RNAi. Thus demonstrated, our core idea is that RNA silencing is useful to selectively reduce mutant huntingtin expression and slow or block neuronal dysfunction and death in HD. We hypothesize that selective knockdown of mutant huntingtin restores normal neuronal function, but excessive silencing impairs neuronal function by interfering with essential signaling events. Aim 1 examines in vivo in mice whether allele specific silencing can delay or prevent HD neuropathy and abnormal behaviors. Aim 2 investigates in post-mortem HD brain post- transcriptional regulation of huntingtin mRNA, to find molecular processes that favor synthesis of mutant huntingtin mRNA. We will use a new strategy to quantify each huntingtin allelic mRNA, based on SNP heterozygosities of the alleles. Knowledge from aims 1 and 2 is essential in applying mRNA silencing to HD patients. Aim 3 studies cellular mechanisms by which RNAi restores essential neuronal signaling activity in HD cells and the threshold for wild type huntingtin to main normal neuronal integrity. Aim 3 combines allelic RNAi and neuronal signaling of the cysteine transporter that regulates glutathione clearance of reactive oxygen species. Aim 4 examines the safety of administration of allele specific siRNA, by evaluating innate immunity and inflammatory responses. This proposal satisfies NINDS goals in translational science: translation of gene silencing therapeutics, early-state therapy development, and identifying mechanisms that underlie nervous system function. Innovations include targeting mRNA alleles for RNAi, use of HD mouse models that express only human huntingtin genes, quantitative measurement of huntingtin allelic mRNA based on SNP heterozygosities, deep sequencing analysis to identify 3 UTR huntingtin mRNA regulation, and zinc finger nuclease strategy to eliminate huntingtin alleles at the genomic level. This proposal applies fundamental biology to understand pathogenesis of HD and translate this knowledge into better therapies.